Helsingin yliopisto Elintarvike- ja ravitsemustieteiden osasto University of Helsinki Department of Food and Nutrition EKT-sarja 1885 EKT-series 1885 Occurrence of vicine and convicine in faba bean and their removal by hydrolysis Marjo Pulkkinen ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry, University of Helsinki, for public examination in lecture hall B3, Viikki, on June 7th 2019, at 12 noon. Helsinki 2019 Custos: Professor Vieno Piironen Department of Food and Nutrition University of Helsinki, Helsinki, Finland Supervisors: Professor Vieno Piironen Department of Food and Nutrition University of Helsinki, Helsinki, Finland Docent Anna-Maija Lampi Department of Food and Nutrition University of Helsinki, Helsinki, Finland Reviewers: Dr Juana Frías Institute of Food Science, Technology and Nutrition, The Spanish National Research Council (ICTAN-CSIC) Madrid, Spain Principal scientist, Dr Anne Pihlanto Natural Resources Institute Finland (Luke) Jokioinen, Finland Opponent: Associate professor Michael Murkovic Institute of Biochemistry Graz University of Technology Graz, Austria ISBN 978-951-51-5270-1 (paperback) ISBN 978-951-51-5271-8 (PDF; http://ethesis.helsinki.fi) ISSN 0355-1180 Unigrafia Helsinki 2019 Abstract Legumes are a sustainable source of plant protein, and their production could be increased in Europe. The use of faba bean (Vicia faba L.) is limited in part due to the presence of the pyrimidine glycosides vicine and convicine. Vicine and convicine, and particularly their aglycones, can cause a form of haemolytic anaemia called favism in individuals who have genetic deficiency in the glucose-6-phosphate dehydrogenase (G6PD) enzyme. Different processing methods have reduced the vicine and convicine contents to varying levels, but the formation of the aglycones have not been studied. Practical processing methods for removal of vicine, convicine and their aglycones are still required. The main aim of this study was to implement methods for the total elimination of vicine and convicine, with a focus on the aglycones. The compounds of interest were quantified using reversed phase high performance liquid chromatography (RP-HPLC) with ultraviolet (UV) detection against an internal standard uridine. The contents of vicine and convicine were determined in cultivars grown in Finland, and the contents were compared within a selected growing year and among three growing years. Vicine and convicine were hydrolysed in faba bean extracts, in vicine and convicine fractions and in faba bean suspensions by using ȕ- glucosidase to study the formation and stability of the aglycones. Finally, the formation and stability reactions were studied under selected model conditions and in sourdoughs and breads. The performance of the HPLC method was suitable for the analysis of vicine and convicine. The levels in the studied cultivars varied from 5.2–7.6 mg/g dry matter (DM) and 2.1–3.6 mg/g DM within one growing year for vicine and convicine, respectively. Cultivar comparison showed that the cultivar ‘Kontu’, the commonly grown cultivar in Finland, contained high amounts of vicine and convicine. No extensive variation was noted among three studied growing years, even though the weather conditions varied markedly. The aglycones were detected and monitored with the HPLC method used for vicine and convicine analysis. The aglycones formed in the vicine and convicine fractions decreased in amount and finally lost their UV absorptivity after 2 h at pH 5 at 37 °C. The need for an external enzyme source was confirmed, as losses of vicine and convicine were rather small in faba bean suspensions. Selected lactic acid bacteria (LAB) strains were able to hydrolyse vicine and convicine in faba bean sourdoughs at 25 °C, 24 h; the hydrolysis depended on the fermentation temperature. Sourdoughs lost up to 82–85% of the vicine and up to 34– 47% of the convicine. The amounts of vicine and convicine in wheat breads, containing 30% faba bean, were comparable to the amounts that were in sourdoughs after 24 h of fermentation. The aglycones were measured from sourdoughs fermented at 25 °C and from the corresponding doughs, but not from the breads. This study showed that vicine and convicine can be analysed simultaneously with their aglycones with an RP-HPLC-UV method, which provides the benefit of estimation of the total elimination of these compounds. The aglycones were found to disappear in all the studied matrices. Fermentation can induce losses of vicine and convicine, but the efficiency of hydrolysis depends on the selection of strains and the fermentation conditions. Furthermore, controlled acidification is necessary for maintain acceptable sensory quality. Acknowledgements This study was carried out during years 2013–2019 at Department of Food and Environmental Sciences, later at Department of Food and Nutrition, in the Food Chemistry Division, at University of Helsinki. The work was funded by the Ministry of Agriculture and Forestry (Makera project Fabapapuaines), the August Johannes and Aino Tiura Foundation of Agricultural research, the Finnish Food and Drink Industries’ Federation, the Doctoral Programme in Food Chain and Health, grants from the University of Helsinki and the Finnish Cultural Foundation (from Foundation of A.E. Sandelin). I am grateful for financial support of this work. I owe my deepest gratitude to my supervisors Professor Vieno Piironen and Docent Anna- Maija Lampi. I truly appreciate your excellent supervision, including all helpful discussions we had and guidance you provided. Your enthusiasm towards research has been motivating and inspiring. You made this work possible by your continuous support and encouragement. I am grateful to my co-authors Professor emeritus Hannu Salovaara, Docent Tuula Sontag- Strohm, Docent Frederick Stoddard, Docent Velimatti Ollilainen, Associate professor Kati Katina, Docent Rossana Coda, MSc Jutta Varis, MSc Maheswor Gautam and MSc Xiao Zhou. I am thankful to Miikka Olin for his valuable technical help, and to MSc Zhongqing Jiang for his kind help with beans. It has been a great opportunity to work with all of you, and your expertise and contribution are highly appreciated. I thank Senior Research scientist Juana Frias and Principal scientist Anne Pihlanto for careful pre-examination of this thesis. Thank you for the valuable and helpful comments and suggestions. I want to thank all personnel I have worked with in the former D-building and in the present EE-building for help, shared knowledge and lively working environment. I especially want to thank my present and former colleagues from both Food Chemistry and Grain Technology research groups. Finally, I warmly thank my friends and family for their support and care. Helsinki, May 2019 Marjo Pulkkinen Contents Abstract 3 Acknowledgements 4 List of original publications 7 Abbreviations 8 1 Introduction 9 2 Review of the literature 11 2.1 Vicine and convicine 11 2.1.1 Structure and properties 11 2.1.2 Hydrolysis of vicine and convicine to their aglycones 12 2.1.3 Reactivity of divicine and isouramil 13 2.1.4 Biological effects 16 2.2 Analysis of vicine and convicine and the aglycones 18 2.2.1 Standard preparation 18 2.2.2 Sample preparation 19 2.2.3 Instrumental methods for analysis of vicine and convicine 19 2.2.4 Instrumental methods for analysis of the aglycones 21 2.3 Occurrence of vicine and convicine in faba bean 25 2.3.1 Location in seeds 25 2.3.2 Genetic selection of cultivars 26 2.3.3 Content and variation among cultivars 26 2.4 Removal of vicine and convicine by processing 28 2.4.1 Conventional processing 28 2.4.2 Bioprocessing 29 3 Aims of the study 32 4 Materials and methods 33 4.1 Materials 33 4.1.1 Faba beans 33 4.1.2 Reagents and micro-organisms 34 4.2 Analysis of vicine, convicine and their aglycones 34 4.2.1 Preparation of the convicine standard (I) 34 4.2.2 Extraction of vicine, convicine and their aglycones (I, III) 35 4.2.3 Optimisation and implementation of the HPLC-UV method (I, II, III) 35 4.3 Enzymatic hydrolysis of vicine and convicine to their aglycones 38 4.3.1 Hydrolysis with added ȕ-glucosidase (II) 38 4.3.2 Determination of ȕ-glucosidase activity from faba beans (III) 40 4.3.3 Analysis of vicine and convicine in fermented doughs and breads (III) 40 4.4 Data analysis 42 5 Results 43 5.1 Determination of vicine, convicine and their aglycones (I, II, III) 43 5.1.1 Comparison of the extraction conditions (I) 43 5.1.2 Optimisation of the HPLC-UV method (I) 43 5.1.3 Applicability of the method for analysis of the aglycones and further products (II) 46 5.2 Vicine and convicine in domestic faba beans (I) 48 5.3 Enzymatic hydrolysis of vicine and convicine (II, III) 50 5.3.1 Hydrolysis by added ȕ-glucosidase (II) 50 5.3.2 Vicine and convicine losses in suspensions, sourdoughs and breads (III) 51 5.3.3 Formation and stability of the aglycones (II, III) 56 6 Discussion 61 6.1 Evaluation of the method for analysis of vicine, convicine and their aglycones 61 6.1.1 Extractability of vicine, convicine and their aglycones 61 6.1.2 Performance of the HPLC analysis for vicine and convicine 62 6.1.3 Simultaneous HPLC analysis of the aglycones 63 6.2 Occurrence and variation of vicine and convicine in domestic faba beans 65 6.3 Elimination of vicine and convicine by enzymatic hydrolysis 66 6.3.1 Hydrolysis of vicine and convicine 66 6.3.2 Challenges in removal of vicine and convicine in LAB fermentation 68 6.3.3 Formation and stability of the aglycones in models 70 6.3.4 Formation and stability of the aglycones in food systems 73 6.3.5 Further challenges in evaluation of the aglycone degradation 74 7 Conclusions 76 8 References 78 List of original publications This thesis is based on the following publications: I Pulkkinen M., Gautam M., Lampi A.-M., Ollilainen V., Stoddard F.